Flow cytometry in the study of cell death

In this report we present a concise review concerning the use of flow cytometric methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. The applications of these techniques to clinical and basic research are also considered. The following cell features are useful to characterize the mode of cell death: (1) activation of an endonuclease in apoptotic cells results in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, leads to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content make it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of apoptotic process; (2) plasma membrane integrity, which is lost in necrotic but not in apoptotic cells; (3) the decrease in forward light scatter, paralleled either by no change or an increase in side scatter, represent early changes during apoptosis. The data presented indicate that flow cytometry can be applied to basic research of the molecular and biochemical mechanisms of apoptosis, as well as in the clinical situations, where the ability to monitor early signs of apoptosis in some systems may be predictive for the outcome of some treatment protocols

Early incorporation of polysulphides in sedimentary organic matter

The increase in sulphur content of organic matter with depth in Recent sediments has been attributed to incorporation of either H₂S or polysulphides or to a combination of both. Indeed, the widespread occurrence of organic sulphur compounds with carbon skeletons that bear an unambiguous link with natural precursors indicates that organic matter may act as a sink for inorganic sulphur species with an efficacy second only to reactive iron minerals. Laboratory and field observations indicate that all compounds identified so far are consistent with incorporation of H₂S; molecular evidence for incorporation of polysulphides has previously been lacking. Here we report the identification of homologous series of cyclic disulphides with a linear carbon skeleton and of a cyclic di- and trisulphide with a C₂₀ isoprenoid carbon skeleton in sediments of Quaternary to Pliocene age. Although incorporation of H₂S can still explain the presence of cyclic disulphides, the cyclic trisulphide implies incorporation of inorganic polysulphides in sedimentary organic matter at the earliest stages of diagenesis

Escalated lymphodepletion followed by donor lymphocyte infusion can induce a graft-versus-host response without overwhelming toxicity.

International audienceTreatment of relapse of hematological malignancies following allogeneic hematopoietic SCT (allo-HSCT) remains very challenging and relies usually on the readministration of chemotherapy combined with donor lymphocyte infusion (DLI). To enhance DLI effectiveness, lymphodepletion (LD) with fludarabine (Flu) and/or CY before the injection of lymphocytes is an attractive modality to modify the immune environment, leading possibly to suppression of regulatory T cells (T(reg)) and exposing the patient to cytokine activation. However, LD before DLI may lead to induction of deleterious GVHD. To avoid inducing overwhelming toxicity, we proceeded by escalating doses of both LD and DLI. Eighteen patients with various non-CML hematological malignancies who relapsed following allo-HSCT were treated with chemotherapy and LD-DLI or LD-DLI upfront. T-cell subpopulation and DC levels as well as cytokine plasma levels (IL-7, IL-15) were measured before and following LD-DLI. Cumulative incidence of acute grade II-IV GVHD was 29.4% similar to that reported in patients receiving DLI without LD. In addition, Flu alone with low dose of DLI was not associated with severe GHVD. CY/Flu at the respective doses of 600 mg/m(2) on day 1 and Flu 25 mg/m(2)/day on days 1-3 did not result in a marked decrease of T(reg) cells, nor in endogenous IL-7 and IL-15 production. However, a peripheral expansion of DCs was observed. These findings suggest that the escalated dose procedure appears safe and prevent overwhelming toxicity. A dose-limiting toxicity has not yet been reached.Bone Marrow Transplantation advance online publication, 28 November 2011; doi:10.1038/bmt.2011.231